Background: The significance and influence of organizational culture on Information Technology acceptance, especially in healthcare field, has been recognized as a source of organizational inertia. This study aimed at developing a model of Hospital Information System (HIS) acceptance for non-teaching hospitals of Iran University of Medical Sciences to encourage the authorities to promote organizational culture and successful application of HIS. Methods: The proposed model was developed according to Michigan Organizational Assessment Questionnaire (MOAQ), Harrison, Hofstede models, and Comparative Values Framework (CVF). The questionnaires were designed based on the model and distributed among 400 HIS users in the hospitals under study, who were selected using stratified random sampling. The structural equation modeling method was used for data analysis in LISREL software. Results: According to the final model, the influences of developmental culture on perceived usefulness, the relationship of 4 types of organizational culture with mandatoriness according to CVF, and the relationships of hierarchical and developmental culture with system use were attested. The relationships between supervision and 4 variables of HIS acceptance were confirmed. Furthermore, the influence of process/ result oriented culture on user satisfaction was demonstrated. The normed chi square index (2.60) revealed that the final model was fitted to the data. The indices were as follow: GFI= 0.95, CFI= 0.97, AGFI= 0.88, RMSEA= 0.064. Conclusion: The components and structural relationships in the model of this study are applicable in the related hospitals, and using this model can promote organizational culture and acceptance of HIS by the users.